Kidney stones are a common clinical problem responsible for significant morbidity and economic costs in excess of 2 billion dollars/year. Despite progress in treatment, the recurrence rate of stones remains high. Interrupting early steps in the formation of kidney stones have the greatest likelihood of successfully preventing recurrence. These early steps include nucleation of stone crystals in tubular fluid and their growth and aggregation within the nephron to a size that can interact with some intrarenal structure. It is the hypothesis of this grant that these processes are primarily determined by specific interactions between stone constituent crystals and macromolecules present in tubular fluid and urine. The overall goal of this project is to advance our understanding of the role of one of these interactions by studying those of the principal urinary inhibitory macromolecule osteopontin/uropontin (OPN) and other macromolecular inhibitors of crystal formation in general in the development of kidney stones. The specific aims are: (1) Determining structural motifs of OPN responsible for effects on nucleation, growth, aggregation of calcium oxalate and hydroxyapatite (crystal formation), specifically, (a) the contributions of the N-terminal and C-terminal halves of the molecule, (b) the roles of putative Ca-binding sites, and (c) the effects of post-translational modifications; (2) Characterizing OPN from normals and Ca stone-formers with respect to its effects on crystal formation, including, (a) isolation and biochemical characterization of OPN from urine of normals and stone-formers, and (b) determining whether OPN from Ca-stone formers inhibits stone crystal formation to the same degree as OPN from normal individuals; (3) Investigating the physical chemistry of the interaction of OPN and other macromolecules and stone crystals, by determining (a) the quantitative relationship between macromolecules and growing crystal nuclei, (b) the relationship between their structure and crystal formation, and (c) the effects of the reactions conditions on the interaction between them and growing crystal nuclei, and by examining (d) the interactions between immobilized macromolecules and crystal formation; (4) Examining the effects of regulating OPN production on an experimental model of CaOx stone disease. Specifically, (a) determining whether pharmacolgic upregulation of OPN production ameliorates crystal deposition in animals given Na oxalate either acutely or chronically, (b) developing an in vivo system for transient renal expression of OPN, and (c) determining whether upregulation of OPN expression protects and down-regulation aggrevates crystal deposition in animals given Na oxalate either acutely or chronically.